| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Lake Highland Prep |
A | Alicea | DeMartino | Parekh | Vidrine | NT | |
| 3 | Tohopekaliga |
A | Cruz | Francois | Richey | Medina | 4:36.32 | 4:36.32 St. Cloud Invitational |
| 4 | Jones |
A | Bryant | Dorns | Hall | Yarn | 4:22.03 | 4:22.03 Bishop Moore - Larry Kelly Invite 2024 %28Regional Qualifier%29 |
| 5 | Oviedo |
A | Atwell | Wahl | Schnoor | Price | 4:13.95 | FHSAA 4A District 3 |
| 6 | Mainland |
A | Rivers | Nixon | Polk | Williams | 4:17.00 | |
| 7 | Winter Park |
A | Arriola | Leon | Guitton | Denney | 4:31.97 | 4:31.97 Charlie Harris Relays |
| 8 | Trinity Prep |
A | Burgos | Patino | Macau | Hennebery | DNS |